1) Molecular machinery regulating protein secretion in the acinar cells of salivary glands In the SGs, the major secretory units are the acini that are formed by pyramidal polarized cells, which form small canaliculi at the apical plasma membrane (APM) where salivary proteins and water are secreted. Proteins destined to secretion are packed in secretory granules (SCGs) that are released into the cytoplasm, and transported to the cell periphery. Here, upon stimulation of the appropriate G protein-coupled receptor (GPCR), the granules fuse with the APM, releasing their content into the lumen of the canaliculi.
Our aim i s to study the molecular machinery regulating the formation of the granules from the TGN and their fusion with the APM. We set up an experimental system aimed at imaging and tracking the secretory granules in the SGs of live animals and based on high-resolution intra-vital microscopy performed on a series of transgenic mouse models expressing selected fluorescently labeled molecules. Among them, a mouse expressing the soluble green fluorescent protein (GFP) in both the sub-mandibular and the parotid glands, enables a clear visualization of both the secretory granules and the APM. We estimated that in resting conditions, the major SGs contain approximately 2500-3000 granules per acinus, most of them accumulated in the sub apical area of the PM. Our analysis on the effect of various agonists of GPCRs has revealed two major differences between in vivo and ex-vivo models: 1) the stimulation of the beta-adrenergic but not the muscarinic receptors, enhances the mobility of the SCGs promoting their docking and subsequent fusion at the APM and 2) muscarinic receptors do not play any synergistic role with the adrenergic receptor during exocytosis. Furthermore, by using another mouse model, which expresses the Tomato fluorescent protein fused with a di-palmitoylated peptide, a well-established marker for the plasma membrane, we discovered that the SCGs after fusing with the plasma membrane completely collapse within 30-40 seconds. This result underscores another major differences between in vivo and ex-vivo models in which compound exocytosis (i.e. the sequential fusion of strings of SCGs), has been described as the primary modality of fusion. Notably, we also showed that the granules in close proximity of the APM recruit a series of cytosolic proteins such as actin, suggesting a role for the cytoskeleton during granule exocytosis. To address this issue we transduced the SGs of live rats with the small peptide Lifeact fused with GFP, a novel tool to label dynamically F-actin. Strikingly, we determined that actin is recruited onto the surface of the granules only after fusion has occurred, and it is released in to the cytoplasm only after their complete collapse. The impairment of the dynamics of the actin cytoskeleton, using pharmacological agents such as cytochalasin D (cyto D) or latrunculin A (lat A), did not affect the fusion of the SCGs with the APM, but it blocked substantially their collapse leading to the accumulation of fused granules which often expanded in size. Finally, we found that myosin IIa and IIb, two actin-based motor proteins are recruited on the fused SCGs and that their motor activity is required to drive the gradual collapse of the granules. These results suggest that the acto-myosin complex provides a contractile scaffold around the SCGs that facilitates the completion of the fusion at the APM. This machinery is utilized by other exocrine glands in which the gradual collapse of the SCGs is not energetically favored due to geometrical constrains. 2) Molecular machinery regulating endocytosis in salivary glands of live animals The role of endocytosis in the physiology of the SGs has never been elucidated. Uptake of proteins from either the apical or the basolateral domain of the ductal system have been described but never thoroughly investigated due to the lack of an appropriate experimental model. The presence under physiological conditions of salivary proteins in the bloodstream and of serum proteins in the saliva, argues strongly in favor of a constant and bi-directional transcytotic movement of proteins across the salivary gland epithelium.
Our aim i s to first define the endocytic pathways operating in SGs in vivo, and then to investigate the contribution of the endocytic events in the patho-physiology of the glands and especially during secretion. We set up various experimental systems in the SGs of live rodents aimed at imaging and studying endocytic events, which occur at either the apical or the basolateral membrane of the SGs epithelium, and in stromal cells. Interestingly, endocytosis in the SGs epithelium of a live animal is significantly reduced when compared with cell cultures, whereas endocytosis in stromal cells appears to occur at a faster rate than in vitro systems. Furthermore, we found that stimulation of the secretory activity of the SGs enhances endocytosis from the apical pole whereas does not have any effect on basolateral endocytosis or on uptake from stromal cells. Using both fluid-phase markers, such as fluorescently-labeled dextrans or smaller molecules, and probes that selectively label the PM, we found that in resting conditions the endocytic activity at the APM is extremely low, whereas stimulation of protein but not water secretion enhances internalization via compensatory endocytosis. Since the SGs are target organs for gene therapy we sought to investigate the endocytosis of plasmid DNA. By using a combination of pharmacological inhibitors, immunocytochemistry and IVM, we found that DNA is internalized in all the components of the SGs epithelium (intercalated ducts, large ducts and acini) without utilizing any of the canonical endocytic pathways described so far. Moreover, only a small proportion of internalized DNA is localized in the early endosomal compartments suggesting that lysosomal degradation is bypassed via endosomal escape. Finally, we observed that stimulation of protein secretion enhances the uptake of the DNA in acinar cells by the same compensatory endocytic pathway observed before. These results revealed unconventional endocytic pathways in live animals that may be exploited to better design non viral-based gene therapy. Endocytosis from the basolateral membrane of the SG epithelium was assessed by two main strategies by injecting fluorescently-labeled probes either systemically or directly into the SGs (fluid-phase endocytosis and non-selective membrane internalization. To characterize the nature of the endocytic pathways, we delivered to the SGs probes of different molecular weights (from 0.3 kDa up to 230 kDa) including dextrans, selected polypeptides, and hydrophobic molecules. Although at a slow rate, only small hydrophobic molecules, such as FM1-43 and DiI were endocytosed in the epithelium. All the other molecules were retained in the extracellular space without any evidence for endocytosis in acinar or ductal cells under both resting and stimulated conditions. These results suggest that the basement membrane surrounding the epithelial cells may act as a major barrier to control the access to the basolateral side of the epithelium. Indeed, under conditions in which the integrity of the basement membrane is compromised, most of the delivered molecules were internalized in the epithelium and accumulated in the endosomal compartment. These results highlight a major role of the basement membrane in controlling the accessibility of molecules to the basolateral side of the epithelium, a factor that needs to be taken in consideration when designing drugs that target plasma membrane receptors